A highly specific synaptic mislocalization and degradation of
Synaptotagmin occurs in stoned mutants. Overexpression of Synaptotagmin rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Overexpression of Synaptotagmin I in
otherwise wild-type animals results in increased synaptic dye uptake, indicating
that Synaptotagmin directly regulates the cycling synaptic vesicle
pool size. In vitro interaction studies indicate a physical interaction between Stoned B and Synaptotagmin. The
Stoned A protein is also found in association with vesicles, and it too exhibits an in vitro association with Synaptotagmin. However, the bulk of Stoned A is in a nonmembranous fraction. These studies suggest that Stoned proteins regulate the AP2-Synaptotagmin
interaction during synaptic vesicle endocytosis. It has been concluded that Stoned
proteins control synaptic transmission strength by mediating the retrieval of
Synaptotagmin from the plasma membrane (Fergestad, 2001; Stimson, 2001, and Phillips, 2000).

Since the evidence for physical interaction between Stoned proteins and Synaptotagmin has now gained acceptence, it is interesting to look back at the genetic evidence for an involvement of Stoned proteins in the recycling of Synaptogamin. This genetic evidence consists of an examination of the effects of stoned mutation on the localization of Synaptogamin, carried out by Fergestad (1999), and described in detail below. These results suggest that the Stoned proteins are
essential for the recycling of synaptic vesicle membrane and are required for
the proper sorting of Synaptotagmin during endocytosis.

Several lethal stoned alleles have been identified,
including two transposable element insertions,
stn13-120 and
stnPH1, that lie in the StnA and StnB
reading frames, respectively, and an EMS-induced allele,
stnR9-10, that has not been characterized
at the molecular level. All of these lethal
stoned mutants die as mature embryos after a failure to
hatch from the egg case, apparently from lack of coordinated movement.
This defect is not a result of alterations in gross embryonic
morphology; mutant embryos show normal segmental patterning of the
epidermis, muscles, and nervous system. Thus, the
mutant embryos appear morphologically normal but are impaired in the
ability to move in a coordinated manner (Fergestad, 1999 and references therein).

Wild-type and mutant embryos (22-24 hr) were labeled with each of the
Stoned antibodies to determine the effect of each mutation on protein
expression and localization. At
the embryonic wild-type NMJ, StnA and StnB proteins are highly
concentrated in presynaptic boutons. None of the stoned
mutant alleles display detectable StnB staining in the synaptic
terminal, including the viable stnC
embryo and the stnC third instar larva.
In contrast, the mutants show variable levels of StnA expression. Both
the stn13-120 and stnR9-10 alleles have severely reduced or undetectable levels of StnA expression, whereas the
stnPH1 allele appears to have only
moderately reduced levels of StnA. The viable
stnC mutant NMJ also shows strongly
reduced or undetectable StnA staining at 22 hr AF; however these animals do display very weak StnA expression at the third instar NMJ. These results
suggest that a mutation in the first open reading frame of the
dicistronic stoned locus (StnA) renders the second reading frame (StnB) unreadable. Thus, the stnPH1
allele primarily removes the StnB product, consistent with the transposable element insertion in the second reading frame (StnB), whereas all other alleles strongly effect the expression of both StnA and StnB (Fergestad, 1999).

Using a number of immunological markers, stoned mutant
NMJs appear morphologically and molecularly similar to those of
wild-type, although the terminals appear slightly smaller than normal. Despite this slight structural difference, the mutant NMJs display clear, punctate expression of several vesicle-associated proteins in the synaptic boutons, including CSP and Syb. In addition, the expression of other synaptic
markers, including a neuronal membrane marker (HRP), syntaxin (Syx), and
Rab3, appear normal in the mutant terminals. The
quantified expression level and bouton localization of the synaptic
proteins are similar to those of wild type (Fergestad, 1999).

In contrast, Synaptotagmin (Syt) protein appears to be strikingly mislocalized in all four stoned mutant alleles. Instead of
the punctate bouton localization of Syt observed in wild type, the stoned mutants display reduced Syt expression in the boutons
and the protein aberrantly dispersed throughout the presynaptic
terminal. This mislocalization is not resolved
with development in the viable stnC
allele because Syt expression remains mislocalized at the third instar
NMJ. Double-labeling assays with other presynaptic markers indicate this mislocalization is specific to Syt, because CSP, the neuronal membrane marker HRP, another synaptic vesicle protein (Syb), and the membrane protein Syx display
normal patterns of expression in all mutants. These results
suggest that stoned mutants specifically mislocalize the SV (synaptic vesicle) protein Syt in the synaptic terminal and retain other synaptic proteins properly (Fergestad, 1999).

For all four mutant alleles, the intensity of the Syt and CSP
expression in individual, double-labeled boutons is quantified using
digital confocal imaging and compared with wild-type expression levels
in parallel trials. All mutants show
an equal and similar ~40%-60% loss of Syt synaptic localization,
compared with the normal expression and localization of CSP. This
reduction in Syt expression is significant in all four alleles, and
the alleles are not significantly different from each other. Syt expression in whole embryos was analyzed to
determine further the nature of the synaptic loss of Syt staining.
Quantified Western blots were performed to determine Syt expression
levels in the different stoned alleles. Mutants display a significant decrease in Syt levels, with protein levels in the range of 20%-60% of those found in wild-type embryos. The decrease in in situ
synaptic localization is consistent with the loss of Syt displayed on
Western blots. These findings suggest that Syt is not only mislocalized in
stoned mutants but may also be subject to rapid degradation
when not properly localized (Fergestad, 1999).

To determine whether the striking synaptic staining pattern for
the stoned proteins is consistent with a physiological function at the
synapse, transmission properties were assayed with electrophysiological recordings at the embryonic NMJ. In all stoned mutant
alleles, nerve stimulation produces muscle contraction, demonstrating
that presynaptic depolarization evokes transmitter release and that the
muscle excitation-secretion response is intact. However, evoked excitatory junctional currents (EJC) peak amplitudes are significantly reduced below wild-type levels, typically by 30%-50%, for all stoned alleles. Furthermore, the release of neurotransmitter at mutant synapses is
markedly asynchronous. The asynchronous mutant transmission seems to result from delayed presynaptic vesicle fusion, similar to that observed for the previously identified synaptotagmin mutant (Fergestad, 1999).

The reduced and erratic evoked transmission of stoned
mutants is further increased after prolonged, repetitive stimulation at moderate or high frequencies (5-20 Hz). To determine the nature of
this debilitation, animals were subjected to a high-frequency stimulus
protocol (10 Hz) sustained over a 5 min period. The average EJC amplitude at wild-type NMJs decreases by an absolute amount similar to that of
mutant animals (~500 pA), suggesting that the NMJ of both
genotypes is fatiguing comparably. However, the stoned
mutants start with significantly impaired performance and fatigue by an
average of 50-75% during the stimulus train, whereas wild-type
amplitude decreases by only ~20%-25%. The decrease in mean EJC amplitude is accompanied by a large increase in transmission failure in the stoned synapses. Wild-type synapses maintain high-amplitude, high-fidelity transmission over a sustained stimulation period of 5 min and show no failures even at the end of the stimulus train. In contrast, the failure
frequency of the mutant synapses is initially significant (~5%-10%)
and increases rapidly during sustained stimulation to a level of
25%-80% at the end of the stimulus train. The three alleles that more strongly affect both StnA and StnB expression (stnR9-10, stn13-120, and stnC) show a more marked transmission failure rate (50%-80%) than does the primarily StnB mutant
(stnPH1; 25%). The striking increase in failure rate during a prolonged stimuli train suggests that repetitive stimulation results in a severe depletion of SVs in the stoned mutants (Fergestad, 1999).

The Drosophila embryonic NMJ is characterized by the
presence of presynaptic varicosities (boutons) containing specialized, densely staining, T-shaped structures (t-bars) at the presynaptic active zones,
the putative SV fusion sites. The pre- and post-synaptic membranes surrounding the
t-bars are densely stained and are separated by a cleft ~15 nm wide. Clear SVs of 30-40 nm diameter are observed clustered around the
t-bars in a semicircular area with a radius of ~250 nm. Synaptic vesicles dock with the membrane immediately adjacent to t-bars in preparation for evoked fusion. For analytical purposes, vesicles are considered docked if distributed less than one
vesicle diameter (<30 nm) from the plasma membrane at active sites. Synaptic vesicles also appear outside the clusters
surrounding t-bars, although at a much lower density than that of the
clustered vesicles. Other membrane structures, such as large dense core vesicles and translucent vesicles larger than typical SVs (presumed to be
endosomes), are also occasionally observed in sections through boutons
containing t-bars (Fergestad, 1999 and references therein).

Striking differences in synaptic ultrastructure are observed for all of
the stoned mutant alleles relative to wild-type controls. Presynaptic boutons containing t-bars are present in all
stoned alleles, and the association of presynaptic tissue
with muscle cells is similar to that in wild type, indicating
that development of NMJs in the mutant embryos occurs normally.
However, a striking reduction in the number and density of SVs present in boutons is observed in stoned mutants. All four
stoned alleles have ~50% fewer SVs clustered around the
active zone t-bars, and SV density outside of the
clustered radius surrounding t-bars is also reduced by ~50%. Analysis of stnPH1/Df(1)HM430 animals showed that they displayed features identical to those of animals hemizygous for stnPH1. A similar 50%
reduction in the number of docked vesicles per t-bar is observed at
stn13-120, stnR9-10, and
stnPH1 synapses, whereas the viable
stnC allele has ~30% fewer docked
vesicles per t-bar than does wild type. Thus, SV density is
severely reduced in all mutant alleles, throughout the presynaptic
bouton, at active zones, and in the number of docked vesicles (Fergestad, 1999).

An additional difference observed in stoned bouton ultrastructure is an increase in intermediates of the SV cycle. In wild-type embryos, SVs are the most prominent membrane structures in sections through boutons containing t-bars. A much smaller number of translucent vesicles (cisternae) noticeably larger than SVs and early endosomes are also present in these sections, and, rarely, multivesicular bodies (MVBs) are seen. The early endosomes represent a step in the normal synthetic pathway for SVs in these terminals. Increased numbers of MVBs result from increased synaptic activity. These MVBs are usually removed from the region of the active zone and are targeted to somatic lysosomes. Because MVBs have been shown to contain SV proteins, they represent the normal degradative route for synaptic proteins (Fergestad, 1999).

In stoned mutants, sections through boutons containing
t-bars have a significantly greater number of large vesicles (>60 nm; endosomes and cisternae) than do wild-type synapses. In addition, a greater number of these large vesicles appear within the SV cluster surrounding
active zones in mutant embryos. The number of large vesicles present
within the clustered radius of t-bars is at least three times greater
in all of the mutant strains than in wild type. Furthermore, a significant increase in the number of MVBs is observed in sections through boutons containing t-bars in stoned mutants. stnPH1 and stnC have an approximate fourfold increase in the number of MVBs compared with that in wild type (Fergestad, 1999).

These results indicate that the stoned mutants have normal
gross synaptic morphology; however, these mutants display a severe reduction in synaptic vesicle number and an increase in recycling intermediates including large cisternae and MVBs. This ultrastructural analysis combined with the abnormal labeling of synaptotagmin and
debilitated synaptic transmission strongly suggests a role for the
stoned proteins in regulating the synaptic vesicle-recycling pathways.

Early models of synaptic membrane recycling suggested that newly
endocytosed vesicles join a sorting endosome compartment before
subsequent budding and maturation. In addition, under periods of sustained or high-frequency transmission, large patches of membrane may be retrieved
from the plasma membrane to form an early endosome from which new
vesicles may be generated. Recent studies provide evidence that synapses may also recycle synaptic vesicle membrane directly, without first fusing with an endosomal-sorting compartment. Evidence from
Drosophila argues that these two recycling pathways may act
in parallel, corresponding to functionally and spatially distinct
vesicle pools. The stoned mutants are clearly defective in one or more of
these recycling pathways. All alleles show a significant decrease in
SVs at active zones and throughout the presynaptic terminal and a
correspondingly increased sequestering of membrane in enlarged membranous compartments. These defects may result from a direct defect
in endocytosis leading to improper regulation of vesicle size, or alternatively, the large vesicles may be endosomal compartments that accumulate owing to
an inability to bud and segregate new SVs during endosome-mediated
recycling. The existence of a
population of very large-amplitude spontaneous fusion events (200-500
pA) at stoned synapses suggests that these large vesicles
can function to release neurotransmitter in a constitutive manner
(similar to large dense-core vesicles). However, the apparent
functional competence of these vesicles does not allow for a determination of
whether they represent abnormally large SVs or endosomes that have
spilled over into the active zone. The accumulation of MVBs may be more informative. These structures clearly derive from endosomes and represent a normal degradative pathway in which SV proteins and membrane are targeted to somatic lysosomes. In parallel with MVB accumulation, Syt
levels at the terminal and throughout the embryo decrease by ~50% in
stoned mutants. It is therefore suggested that the Stoned proteins are specifically involved in the recruitment and localization of Syt during SV recycling and, in the absence of correct targeting, that Syt is degraded via a default degradative pathway involving MVBs (Fergestad, 1999).

Mature synaptic vesicles have a specific complement of proteins
required for a variety of functions. Each protein must be selectively
recruited to the maturing SV during endocytosis. The mislocalization of
Syt in stoned synapses is not accompanied by a loss of other
SV proteins (e.g., synaptobrevin and CSP), suggesting that the stoned
proteins may be involved in the specific recycling of Syt from
endocytosed membrane. It is hypothesized that the Stoned proteins normally
function at a choice point segregating recycled Syt protein into
maturing synaptic SVs and away from the MVB degradative pathway. Such a role has been suggested for the AP3 complex, shown recently to be required for localization of a SV transporter protein and to be required for the generation of SVs. Similarly, the Drosophila gene LAP, which encodes AP180 (associated with clathrin-dependent endocytosis with the AP2 complex), has been shown recently to be involved in regulating SV size and the proper recruitment of the vesicle coat protein clathrin (Zhang, 1998). In the C. elegans AP180 mutant (UNC-11), the protein also seems to have a specific role in recruiting synaptobrevin to the recycled vesicle (E. Jorgensen, personal communication to Fergestad, 1999). These AP180 data, combined with the observation that SV size is not altered in mutant animals lacking synaptobrevin, suggest that AP180 has two distinct functions: structural budding of membrane and the specific recycling of synaptobrevin (Fergestad, 1999).

The similarities between these studies led to the hypothesis that
there may be separate mechanisms required for the recycling of each
distinct SV protein and that these mechanisms may be intimately integrated into the membrane-budding machinery. Such a coupled mechanism would guarantee that newly generated SVs have the correct functional complement of SV proteins. Clearly, within this general mechanism, the Stoned proteins couple the specific recruitment of Syt
to proper SV biogenesis. The Stoned proteins may participate in the
AP2-mediated plasma membrane mechanism or, alternatively, act in a
separate and/or later site such as AP3-mediated endosomal sorting to
direct the recruitment and/or localization of Syt into mature SVs (Fergestad, 1999).

Retrieval of SV components from the plasma membrane is tightly
temporally coupled to exocytosis. Mutation of stoned
disrupts this temporal coupling and delays the onset and rate of
endocytosis after exocytosis. Delayed application of FM1-43 to
stoned synapses after stimulation reveals that the delayed
component of endocytosis is comparable with wild-type levels. There are
several possible explanations for these altered endocytosis kinetics in
stoned mutants. A likely rationale is that global membrane
retrieval is delayed in stoned mutants, suggesting that
Stoned proteins may play a role in either initiating endocytosis or
facilitating the speed of endocytosis. An alternative scenario may be
that there are multiple pathways for SV endocytosis, which differ in
temporal kinetics, and that the rapid endocytosis mechanism is
specifically impaired in stoned mutants. Although it is
formally possible that delays in exocytosis might also explain the
delayed membrane retrieval, recordings of synaptic
transmission show that exocytosis speed is only very minimally impaired in stoned mutants (Fergestad, 2001).

How are the numerous different components of the SV recognized and
recombined in the precise stoichiometric ratios required for synaptic
function? There is an increasing body of evidence that a cast of
specific recycling proteins is required to recognize and retrieve
specific components of the SV during plasma membrane endocytosis. At
center stage, the AP2 complex plays a prominent role in
clathrin-mediated SV endocytosis. Genetic removal of the alpha-Adaptin subunit in
Drosophila shows that the AP2 complex is absolutely required for the SV endocytotic process in this system (Gonzalez-Gaitan, 1997). Moreover, the AP2 complex has been shown to bind
Synaptotagmin (Zhang, 1994), and it thus seems likely that
this complex mediates the endocytotic recovery of Synaptotagmin, likely
in addition to other integral SV proteins (Fergestad, 2001).

It is proposed that the role of the Stoned proteins may be to recycle
Synaptotagmin by mediating the association with the AP2 complex. It
has been shown recently that both StnA and StnB bind with high
specificity to Synaptotagmin (Phillips, 2000) and therefore
likely act in a cooperative manner in Synaptotagmin retrieval.
Studies reported by Haucke and De Camilli (1999) have shown that the
AP2-Synaptotagmin interaction can be stimulated by the presence of
Yxxphi-containing peptides, which also enhance the recruitment of AP2
to the plasma membrane. Because both Stoned proteins contain multiple
copies of these tyrosine-based motifs, as well as other AP2 and
Clathrin binding domains, it is hypothesized that the Stoned
proteins specifically promote the retrieval of Synaptotagmin from the
plasma membrane by mediating the AP2-Synaptotagmin binding. Because
the Stoned proteins are encoded by a dicistronic locus, polarity
constraints have to date prevented an independent dissection of the
roles of StnA and StnB in Synaptotagmin endocytosis. Why does the
process require two proteins, and what does each contribute to the
recycling mechanism? Targeted homologous knock-out techniques are being
used to explore these questions (Fergestad, 2001).

As the proposed calcium sensor for SV fusion, Synaptotagmin is likely
to function as a key regulator of transmission strength. The number of
Synaptotagmin proteins in an SV membrane may play an important role in
regulating the response of the presynaptic terminal to depolarizing
stimuli. Overexpression of Synaptotagmin alone is capable of substantially
increasing the size of the endo-exo SV pool. Therefore, the Stoned
proteins, by regulating Synaptotagmin recycling, also act as key
regulators of neurotransmission strength. Future experiments are
focusing on the specific regulation of Synaptotagmin levels by each
of the Stoned proteins (Fergestad, 2001 and references therein).

Flies with defects at the stoned locus have abnormal behavior and altered
synaptic transmission. Genetic interactions, in particular with the shibire (dynamin) mutation, indicate a presynaptic function for Stoned and suggest an involvement in vesicle cycling. Immunological studies have revealed colocalization of the Stoned proteins at the neuromuscular junction with the integral synaptic vesicle protein Synaptotagmin (Syt). Stoned interacts
genetically with synaptotagmin to produce a lethal phenotype. The StnB protein is found by co-immunoprecipitation to be associated with synaptic vesicles, and glutathione S-transferase pull-downs demonstrate an in vitro interaction between the micro2-homology domain of StnB and the C2B domain of the SytI isoform. The StnA protein is also found in association with vesicles, and it too exhibits an in vitro association with SytI. However, the bulk of StnA is in a
nonmembranous fraction. By using the shibire mutant to block endocytosis, StnB has been shown to be present on some synaptic vesicles before exocytosis. However,
StnB is not associated with all synaptic vesicles. It is hypothesized that StnB
specifies a subset of synaptic vesicles with a role in the synaptic vesicle
cycle that has yet to be determined (Phillips, 2000).

Hypomorphic mutations at the Drosophila synaptotagmin locus result in behavioral, electrophysiological, and morphological phenotypes similar to those seen in stn mutants. Synaptotagmin (Syt) is a synaptic vesicle
protein with a proposed role in both exocytotic and endocytotic functions in both mammals. The aim of these experiments was to determine whether either of
the viable stn alleles, when in combination with mutations at the synaptotagmin locus, enhances or suppresses the
syt phenotype. The stnC and
stnts alleles are homozygous
viable as adult flies and were isolated in the same genetic background,
and both possess the same insertional polymorphism as the original
Oregon-R strain. The flies used were heterozygous for two
stn null mutations, sytAD4 and
sytD27. The P-element-mediated transposition of a stn minigene to the third chromosome provides 10% of wild-type Syt protein levels and
allows the survival of flies with lethal null mutations on both chromosomes at the
syt locus to produce fertile adults. Double mutant combinations
were constructed, and the viability and behavior of the resulting flies were investigated. The data clearly indicate a genetic interaction between the
stnts mutations and syt. Because synaptotagmins are an integral component of synaptic
vesicle membranes, this data also suggests an interaction between the
stoned protein(s) and synaptic vesicles (Phillips, 2000).

Hydrophobicity analysis of both the StnA and StnB proteins
suggests that they should be soluble proteins. Wild-type fly head extracts, homogenized in both the presence and absence of calcium, were
subjected to differential centrifugation to produce P1 (1000 × g), P2 (25,000 × g), and P3 (125,000 × g) pellets and a final supernatant fraction, S3. Western blots prepared from these fractions were probed with anti-StnA, anti-StnB, and anti-Syt antibodies. The
anti-Syt antibodies were raised against the recombinant cytoplasmic
region of the Syt protein and recognize a number of isoforms of
Synaptotagmin (54-69 kDa). Neither the StnA nor StnB proteins could be visualized in the supernatant fraction. The StnB protein co-sediments with the synaptic
vesicle protein marker Syt, primarily in the P2 and P3 fractions. StnA,
in contrast, preferentially partitions into the P1 fraction,
although some StnA was found in both the P2 and P3 fractions. This indicates
that both stoned proteins preferentially partition into
either membrane fractions or fractions containing large protein
complexes. The association of StnA with the P1 fraction was investigated further. Solublization of StnA from P1 was not achieved with Triton X-100, deoxycholate, or high NaCl concentrations; however, the chaotropic agent KI effectively solubilizes all of the StnA protein from the P1 fraction. This indicates that StnA in the P1 fraction is not associated with heavy membranes but is more likely to be associated with a large protein complex (Phillips, 2000).

As expected, the Syt isoforms were associated with both the P2 and P3
fractions, plasma membrane, and vesicle-enriched fractions, respectively. Also observed was a coincidental shift of Syt and StnB
from the P3 to the P2 fraction when homogenization was performed in the
presence of Ca2+. The supernatant fraction
from the P1 centrifugation (S1) was applied to a glycerol gradient and
centrifuged to separate membrane components. A peak of Syt,
corresponding to the synaptic vesicle fraction, was observed. StnB protein
co-sediments with the Syt peak, whereas StnA, although entering the
gradient, peaks in fractions 3-6. These
two results, the coincident redistribution of StnB and Syt in the
presence of Ca2+ and their
co-sedimentation in glycerol gradients, are consistent with an
association of the StnB protein with synaptic vesicles. The
plasma membrane marker syntaxin was also present in the gradients, probably indicating fragmentation of plasma membrane during
homogenization, although its distribution does not mirror that of
synaptotagmin/StnB or StnA (Phillips, 2000).

To determine whether the StnB present in the P3 fraction is associated
with synaptic vesicles, anti-StnB antibodies were attached to Protein
A-coated magnetic beads, and incubated with a P3 fraction prepared from
Drosophila head homogenates in the absence of calcium. These
beads were then analyzed for the presence of StnB and the synaptic
vesicle protein markers Syt, Csp, and Syb as well as the plasma
membrane marker Syx. The results indicate that a major proportion of
the StnB protein in the P3 fraction is immunoprecipitated. The StnB antibodies
coprecipitate all three synaptic vesicle markers (Syt, Csp, and Syb),
but not Syx. Although multiple species of Syt can
be identified in P3 fractions, only the 69 kDa Syt isoform is
present in these precipitates (Phillips, 2000).

On the basis of its deduced amino acid sequence, StnB does not contain
any putative transmembrane segments and is unlikely to be an integral
membrane protein. What then is the molecular nature of the StnB/vesicle
association? To address this question, fractions of the StnB
immunoprecipitations were washed extensively with 1% Triton X-100. The
presence of the detergent entirely removes Csp from the precipitates
and considerably reduces the amount of Syb present. However, Triton
X-100 has no effect on the amount of Syt bound to the beads. This result suggests that StnB is not associating with
the lipid components of the vesicle membrane and that the interaction
may be via Syt (Phillips, 2000).

There was relatively little StnA seen in the P3 fraction on the Western
blots. However, StnA protein was observed on the glycerol gradients, and although
there was no coincidence of the peak fractions, there was overlap
between StnA and the synaptic vesicle peak. The immunoprecipitations
were therefore further probed for the presence of StnA. The StnA
protein was found to be associated with the immunoprecipitates and to
be insensitive to the Triton X-100 washes. When
immunoprecipitations were performed using anti-StnA antibodies attached
to beads, again Syt and Csp were coprecipitated. Therefore, both StnB and StnA can be found associated with synaptic
vesicles in the P3 fraction (Phillips, 2000).

The continued association of Syt with immunoprecipitated StnB
even after Triton X-100 treatment suggests a direct interaction between StnB and Syt. It appeared likely that this interaction would
be via the µ2-like domain of StnB. To investigate this, a 621 residue
protein containing the µ2-like region, residues 883-1089, and
adjacent sequence up to residue 1261
was expressed in the pGEX expression vector to produce a GST fusion.
The resin-bound GST/µ2-like fusion protein was incubated overnight
with a crude extract of bacterial cells expressing the cytoplasmic
portion of Drosophila Syt as a 6x-His (pET) fusion protein
of 39 kDa. When proteins eluted from the resin were Western-blotted and
the blot was exposed to anti-Syt antibody, the expected 39 kDa Syt fusion protein was identified. The GST protein itself was unable to bind Syt in this assay. A series of fusion constructs with different regions of the µ2-like domain of StnB were similarly assayed. In repeated experiments, fusion proteins terminating at amino acid residue 930 were unable to bind Syt. However, Syt binds to all fusion proteins containing amino acid residues 847-1108 of the StnB protein. Because residues 883-1138 of StnB constitute the µ2-like domain, this domain is sufficient for Syt binding in vitro. It is possible, however, that residues outside the µ2-like domain influence the strength of this interaction in vivo (Phillips, 2000).

StnA can be found associated with
synaptic vesicles immunoabsorbed by the StnB antibodies. To determine
whether the StnA protein might be associated directly with
synaptotagmin, Syt binding to StnA fusion proteins was analyzed. Three constructs were used. The first was a fusion protein that included the first 290 residues of StnA (GST/5'StnA), the second included residues 26-350
(GST/StnA Xho-p33), which removes most of the N-terminal region that
would be missing if the methionine in the stnts mutant acted as a novel translation initiation site, and the third was identical to GST/5'StnA
but contained the sequence encoding the K to M substitution found in
the stnts flies. The results indicate
that residues 26-290 of the amino terminal region of StnA can bind
Syt. This region includes the sequence altered in the
stnts mutation. Binding of Syt to the GST/5'StnA construct containing the stnts mutation shows that this
mutant protein also binda Syt. The affinity of StnA for Syt appears less than that seen with the StnB constructs; however, the binding of StnA to Syt in vitro is observed consistently (Phillips, 2000).

There are two C2 domains, C2A and C2B, in the Syt monomer, and
the two domains have been found to interact with different intracellular components. Two Syt protein constructs were produced, each containing one of the C2 domains, and the ability of these protein constructs to bind to both the StnA/GST and StnB/GST fusions was investigated. The protein containing the
cytoplasmic sequences including the C2A domain (residues 137-327) but
excluding the C2B domain failed to bind to either StnA or StnB.
However, the protein containing only the C2B domain (residues 317-473)
was found to bind to both StnA and StnB (Phillips, 2000).

The immunoprecipitation studies indicate that only the 69 kDa Syt isoform is coprecipitated with StnB. However, there are other synaptotagmin species in Drosophila, including a SytV homolog of 55 kDa that are associated with synaptic vesicles. The C2B domain (residues 325-374) of SytV was expressed as a pMAL fusion protein and shown to cross-react with the polyclonal anti-Syt antibodies. Therefore, it was asked whether StnB could interact with SytV in vitro. The SytV fusions were then assayed for their ability to bind to StnB. The SytV C2B domain indeed interacts with the µ2-homology region of StnB (Phillips, 2000).

Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the micro-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. This study report that these interactions are increased in the presence of Ca2+. The Ca2+-dependent interaction between the micro-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound Synaptotagmin-1 associated synaptic vesicles (Soekmadji, 2012).

This study has investigated the effect of Ca2+ upon Stoned proteins binding to SYT-1 C2B domain. Previous study has show the Drosophila μHD of STNB binds to C2B domain of SYT-1, which is in agreement with a study that showed the μHD of the stonin-2, the mammalian homologue of the Drosophila STNB protein, could also bind SYT-1. It has also been reported that stonin-2 is able to bind the C2A domain, even though the role of Ca2+ in this binding was not explored. This study also observed that STNB is able to bind to the C2A domain in vitro, albeit at a much lower level as compared to the binding to the C2B domain. The data show that the binding of Drosophila STNB to Drosophila SYT-1 is different from that of the murine μ2 subunit of AP-2. While μ2 binding to C2B domain requires phosphatidylserine (PS) and Ca2+, the binding of the STNA and STNB proteins did not require PS. In fact PS-containing phospholipids nearly abolished the STNB binding to SYT-1 C2B. Deletion of the polyK region of the C2B domain also did not abolished STNB binding to SYT-1, indicating that this region cannot be the binding site for either STNA or STNB. The data support a recent publication that showed that, in Drosophila, μ2 cannot replace the function of the μHD of STNB in vivo, and suggest that STNB and AP-2 might represent alternative mechanisms for synaptic vesicle recycling (Soekmadji, 2012).

AP-2 is ubiquitously expressed and implicated in general mechanisms of endocytosis from the plasma membrane, while in Drosophila, STNB is expressed and functions only in the nervous system. Differences in the μHD of STNB and the μ2 domain of AP-2 may reflect the need for flexibility of μ2 subunit of AP-2 to act in a number of endocytic pathways, while the function of STNB may be specific for synaptic vesicle retrieval. The data, in conjunction with a report that STNB may specifically regulate the sorting of a subset of SV, suggest the Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+-bound SYT-1 associated SV (Soekmadji, 2012).

Upon Ca2+ binding, SYT-1 was reported to undergo a conformational change that protects SYT-1 against trypsin and chymotrypsin digestion in vitro. Ca2+ has also promoted homo-oligomerization of SYT-1 and/or hetero-oligomerization with other synaptotagmins. Studies using mutations that affect Ca2+ dependent oligomerization, such as Y311N, showed a partial inhibition for internalization of SYT-1 in PC12 cells while the corresponding mutation in Drosophila (AD3) alters the rate of exocytosis. The AD3 SYT-1 is still capable of binding to SNARE complexes, synprint and AP-2, which implies that the mutation does not cause a complete loss of function. Hence the docked vesicles in AD3 flies (a mutation that affects Ca2+ dependent oligomerization) require higher Ca2+ concentration to undergo exocytosis, suggesting that defects in Ca2+ dependent oligomerization renders C2B SYT-1 inefficient for exocytosis. It was reported that Ca2+-triggered SYT-1 clustering is via the C2B domain and is required for exocytosis. In a potential endocytic model that includes STNB, the oligomerized SYT-1 that has led to exocytosis, could then be a target for STNB binding, and STNB, in turn, could recruit dynamin via the intersectin DAP-160. This would create what amounts to a Ca2+-dependent endocytic complex. However, a previous study has shown that STNB can be found bound to synaptic vesicles, via SYT-1, prior to exocytosis, that is, prior to the Ca2+ influx that might trigger SYT-1 oligomerization and the coupling of excitation and vesicle fusion. Either this bound STNB reflects the low level of oligomerization of SYT-1 in the absence of Ca2+, or that other factors, such as those that might alter the structure of the C-terminal region of SYT-I C2B, are playing a role in potentiating the binding of STNB to SYT-1 even in the absence of Ca2+. Certainly there is no STNB protein in the soluble fraction from fly head extracts, suggesting that all STNB is in a bound form, and although some is certainly is, perhaps not all is bound to SYT-1 (Soekmadji, 2012).

The D3,4N mutation in transgenic flies results in a reduction in the rate of endocytosis of synaptic vesicles, this may be due to a failure of this mutant SYT-I to interact with Stoned proteins. Another mutation which gives constitutive dimerization, the D3,4N mutation, was incapable to restore internalization in CHO cells. The D3,4N mutation did not only show a reduction in the rate of endocytosis in Drosophila, but also exocytosis defect by decrease evoked transmitter release and reduce in apparent Ca2+ affinity for synaptic transmission. Indeed, similar to these synaptotagmin mutant flies, the stoned mutants showed alterations in both spontaneous and evoked release at larval NMJ and severe neurotransmission defect that may lead to embryonic lethality, as well as depletion of synaptic vesicle and increase of membrane recycling intermediate that might be due to mislocalization of synaptotagmin during endocytosis. In contrast with μ2 and SYT-1 interaction, the binding of Stoned proteins do not require phospholipids. Studies using Folch liposome has shown that D3,4N mutant are not able to induce a close proximity membrane curvature, which may be a prerequisite for SNARE mediated membrane fusion. A synthesized peptide consist of the 3rd loop of C2B SYT-1 can outcompete the μ-homology domain of STNB, suggesting the interaction of STNB to SYT-1 is mediated by a region in loop 3 of C2B domain. Thus, it is an attractive hypothesis that STNB may act as an inhibitor for membrane fusion. In a recent paper, it is shown that SYT-1 bound to PtdIns at the same level as PS and that this binding is also required Ca2+; while the binding of PIP2 with SYT-1 is less affected by Ca2+. It would be interesting to investigate whether these lipids will have similar effect as PS in affecting Stoned and C2B SYT-1 binding (Soekmadji, 2012).

The role of STNA in the synaptic vesicle cycle remains elusive. The presence of STNA at the larval NMJ appears nonessential. However STNA is certainly associated with synaptic vesicles and it is clear that STNA has a strong affinity for SYT-1. This study has shown that the oligomerization of SYT-1 dramatically increases the binding of STNA and presumably STNA will bind under similar in vivo conditions as STNB. The presence of putative AP-2 binding motifs in STNA, may make vesicles bound by STNA targets for AP-2 mediated endocytosis. This is in contrast to STNB that lacks such AP-2 binding motifs. Whatever the specific mechanism of action of the STNA protein, it is clear from this study that the action of Ca2+ has a marked effect on STNA and STNB association with SYT-1 and confirms them as important proteins in the mechanism(s) of synaptic vesicle recycling in Drosophila (Soekmadji, 2012).

Using antibodies specific for the Stoned proteins, the distribution of StnA and StnB in the nervous system was examined. Both proteins are strikingly expressed at synaptic connections both in the CNS and at the neuromuscular junction in the mature embryo (20-22 hr AEL) and throughout larval development. In the third instar NMJ, both Stoned proteins are highly expressed in all synaptic bouton types, including type I, II, and III boutons. Both StnA and StnB proteins show precise colocalization with presynaptic markers, such as the synaptic vesicle-associated cysteine string protein (CSP), suggesting a presynaptic localization. Double-labeling experiments using antibodies against known postsynaptic proteins, such as the membrane-associated Discs large, show the presynaptic StnA and StnB proteins surrounded by a halo of the postsynaptic marker (Discs large), consistent with restriction of the Stoned proteins to the presynaptic region. Similar results were obtained using a different postsynaptic marker, the GluRII glutamate receptor, which also shows a halo of GluR protein expression surrounding the Stoned labeling. These results suggest that both Stoned A and B are present exclusively at the presynaptic compartment where the proteins colocalize with SV pools (Fergestad, 1999).

Stoned proteins, Synaptotagmin I and plasma membrane endocytosis

StnA and StnB act cooperatively to regulate synaptic vesicle recycling events in Drosophila. Both proteins localize to the presynaptic compartment and occupy common subsynaptic domains. Recent work from a variety of laboratories has precisely defined spatial and functional domains within synaptic boutons at the Drosophila NMJ. These domains include the active zone, periactive zone, membrane-associated and internal vesicular pools, and a well defined 'network' or 'lattice' domain that exclusively localizes endocytotic proteins. The endocytotic domain is of particular interest because of the hypothesis that the Stoned proteins mediate vesicular recycling. Previous studies have shown that alpha-Adaptin, a subunit of the endocytotic AP2 Clathrin-associated adapter complex, and Dynamin, the GTPase 'pinchase' mediating endocytosis, both localize to the highly characteristic lattice occupying the area surrounding the active zone domains (Gonzalez-Gaitan, 1997; Fergestad, 2001).

The localization of StnA and StnB relative to these well defined presynaptic domains has been determined. Both StnA and StnB proteins colocalize tightly with the endocytotic proteins alpha-Adaptin and Dynamin. All four proteins lie within the endocytotic lattice that surrounds but excludes the exocytotic active zones. Like Dynamin, both StnA and StnB are tightly associated with the plasma membrane and do not occupy cytosolic domains in the bouton interior. Markers of the active zone and vesicular pools, such as the SV-associated Cysteine String Protein (Csp), do not colocalize with the Stoned proteins but rather occupy the domains within the endocytotic lattice. This confocal analysis supports the localization of both StnA and StnB proteins with the endocytotic network and not with SV pools and areas of exocytosis (Fergestad, 2001).

The shibireTS1 mutation disrupts Dynamin function and provides a temperature-dependent block in the vesicle-budding step of endocytosis. Stimulation of the Drosophila NMJ in shibireTS1 mutants at the restrictive temperature (30°C) depletes the SV population because SVs are driven into the plasma membrane in the absence of endocytosis. Immunological staining of these vesicle-depleted shibireTS1 terminals shows that SV markers, such as Csp, become associated exclusively with the plasma membrane. shibireTS1 SV-depleted terminals were labeled with antibodies against StnA, StnB, and alpha-Adaptin. No alteration in the endocytotic network, including the distribution of the Stoned proteins, was observed. These studies confirm that both StnA and StnB are associated with the plasma membrane and do not associate with internal vesicles. Returning SV-depleted shibire TS1 terminals to the nonrestrictive temperature (22°C) allows endocytosis to resume, resulting in mass membrane retrieval from the plasma membrane. After SV depletion (30°C for 10 min) and brief recovery to permit massed endocytosis (22°C for 10 min), no detectable alteration in the expression pattern of the endocytotic proteins, including both StnA and StnB is observed. These data support the conclusion that the Stoned proteins occupy only the endocytotic domain within synaptic boutons and are tightly associated with the plasma membrane (Fergestad, 2001).

There is a prominent mislocalization of Synaptotagmin I protein in stoned mutants; stoned function is required to maintain Synaptotagmin I in tight synaptic bouton domains and to prevent its loss throughout the arbor and proximal regions of the axons and its eventual degradation. It was of interest to determine whether this relationship is reciprocal by testing whether the Stoned proteins are mislocalized and/or degraded in the absence of Synaptotagmin. Immunohistochemical studies in sytAD4, a null allele of synaptotagmin, have revealed no detectable alteration in either StnA or StnB expression at the embryonic NMJ. Both Stoned proteins are maintained in tight bouton puncta in the complete absence of Synaptotagmin. These data suggest that the Stoned proteins are specifically required for the recycling of Synaptotagmin but do not require Synaptotagmin for their localization within the endocytotic domains. Furthermore, these data suggest that the phenotypes observed in synaptotagmin mutants do not result from aberrant localization of the Stoned proteins (Fergestad, 2001).

stoned mutants exhibit impaired synaptic transmission and a reduced number of morphologically abnormal SVs, suggesting a defect in vesicular recycling at the synapse. To assay for SV recycling defects directly, the fluorescent lipophilic dye FM1-43 was used in membrane retrieval and SV-recycling assays at the NMJ. Extracellularly applied FM1-43 dye is incorporated into SVs after endocytosis, reliably maintained in SVs and vesicular intermediates, and released during stimulated exocytosis. Incubating Drosophila larval NMJ preparations with FM1-43 in a depolarizing solution of high K+ (90 mM) results in specific dye incorporation in SVs of the presynaptic boutons that can be released after subsequent depolarization and fusion. This analysis was used, in various experimental paradigms, to assay endocytosis and SV recycling in stoned mutant NMJs (Fergestad, 2001).

Wild-type and stoned mutant third instar animals were dissected in the same chamber and stimulated identically with 5 min of high-K+ saline in the presence of FM1-43. Control boutons revealed robust endocytosis and strongly incorporated dye, whereas stoned mutant boutons loaded dye very poorly under the same conditions. The mean density of FM1-43 incorporation in larval NMJ boutons (>5 µm) was quantified and normalized to that of the control. The viable stnC mutant animals display a significant impairment in dye uptake. Examination of two lethal stoned alleles, stn13-120 and stnPH1, reveals an even more profound defect in endocytosis. Because severe stoned mutations are all embryonic lethal, normalized comparisons of FM1-43 dye uptake to control were done at the embryonic NMJ. Wild-type embryonic NMJs can be loaded with FM1-43 by high-K+ depolarization, and all synaptic boutons appear to label, generating a signal comparable with antibody staining against SV proteins at the same stage (21-23 hr AF). In sharp contrast, both lethal stoned alleles show a >90% reduction in dye incorporation, making measurable endocytosis essentially undetectable. These studies show that three different alleles of stoned all show severe defects in exo-endo SV recycling at the NMJ synapse and that the severity of the recycling defect correlates with the severity of the mutant allele examined (Fergestad, 2001).

Although such a dye uptake impairment implies a direct defect in membrane retrieval, the possibility that the reduced exocytosis observed in stoned mutants causes a coupled reduction in endocytosis cannot be excluded. To address this possibility, spaced durations of depolarization stimulation and FM1-43 dye loading were studied. Brief dye loading with high K+ for <1 min (30 sec and 1 min intervals assayed) resulted in a similar defect in stoned dye loading. Dye loading with 5 min of stimulation provided a slight increase in bouton fluorescence intensity in both control and mutant NMJ terminals, but the stoned-specific defect in endocytosis remained unaltered. Furthermore, 10 min of high-K+ application and dye labeling resulted in no further increase in synaptic dye incorporation of either control or stoned mutant animals, suggesting that the cycling SV pool is maximally saturated after <5 min of high-K+ stimulation. Similarly, to determine whether the striking defects in endocytosis in the embryonic lethal mutants were caused by delayed endocytosis, dye was applied for 5 min in calcium-free saline after 5 min of high-K+ stimulation with dye. Longer periods of dye application did not improve the FM1-43 bouton labeling in either wild-type controls or stoned mutants. These studies suggest that the recycling SV pool is saturated at these loading times and that stoned mutants have a specific and severe reduction in SV endocytosis. These findings show that the defect in dye uptake is independent of the stimulation duration and probably results from a smaller recycling pool of SVs in stoned mutants (Fergestad, 2001).

Large NMJ boutons (>3 µm, typically 3-5 µm) in normal Drosophila third instar larvae show characteristic patterns of SV pools. Wild-type boutons loaded with high K+ always incorporate FM1-43 dye in a circular pattern, with the fluorescence restricted to cortical regions underlying the plasma membrane and an absence of signal in the central regions of the bouton. This dye incorporation shows that the recycling SV pool filled via high-K+ stimulation is spatially restricted in a characteristic peripheral ring. In contrast, the bouton interior contains a reserve pool of SVs that are accessed only under conditions of intense transmission demand. The reserve pool can only be loaded with FM1-43 after high-frequency (>30 Hz) stimulation or after complete elimination and mass renewal of the SV population with the Dynamin mutant shibireTS1. It was of interest to determine whether the ready/reserve SV pool boundary is maintained in stoned mutants and whether Stoned proteins may play a role in the spatial dynamics of SV recycling (Fergestad, 2001).

In clear contrast to the normal condition, standard high-K+ FM1-43 labeling in stnC boutons results in the dye filling the entire bouton, including the center of the bouton where reserve pool vesicles are normally located. Recycled vesicles in stoned mutants lack the normal spatial restriction defining the readily releasable and reserve SV pools. This spatial distribution pattern is reminiscent of the FM1-43 signal after dye uptake in the Dynamin mutant shibireTS1. After temperature-dependent depletion of all SVs, shibireTS1 animals return to the permissive temperature undergo mass membrane retrieval coinciding with the formation of early endosomes/cisternae and repopulation of the entire bouton with SVs. shibireTS1 SV dynamics were assayed by first loading the NMJ terminal at the permissive temperature (22°C) and then reloading the same terminal after temperature-dependent (30°C) depletion of all SVs. Before unloading, the shibireTS1 boutons display a labeled circular pool of SVs corresponding to the readily releasable SV pool identical to that of wild-type controls. However, after total SV depletion, shibireTS1 terminals load both ready and internal reserve pools comparable with the pattern observed in stoned mutants. Thus, stoned mutants display aberrant trafficking of newly endocytosed membrane, which may be inappropriately targeted into sorting endosomes in the bouton interior. This conclusion is consistent with the significantly increased incidence of enlarged vesicles and multivesicular bodies observed in stoned mutants at the EM level. This conclusion also supports the hypothesis that loss of stoned function results in increased segregation of membrane and/or protein to the sorting and degradation pathways, at the expense of the recycling SV pool (Fergestad, 2001).

Application of high-K+ saline to FM1-43-labeled synaptic boutons results in a second round of exocytosis that releases the dye contained within the SVs (unloading). Terminals loaded with FM1-43 for 5 min release most of this dye via the fast-cycling SV pool after a comparable 5 min unloading period. Under conditions of equal loading and unloading periods, the majority of dye in both wild-type (85.5 ± 1.0%) and stnC (83.7 ± 5.3%) NMJ synapses is released via Ca2+-dependent exocytosis. In contrast, shortening unloading times to 1 min of high-K+ saline application is still sufficient to unload the majority of dye in wild-type terminals (88.1 ± 1.8%), but the amount of dye released from stoned boutons is significantly reduced. These findings confirm that the readily releasable pool is smaller in stoned mutants, and although these vesicles are competent to fuse, they do so in a slower time course. The impairment of dye release is consistent with the defect in exocytosis observed in stoned mutants and may result from the aberrant vesicle trafficking observed in stoned terminals. These data further suggest that the aberrantly distributed SVs in stnC mutants are releasable and do not have the 'barrier' thought to spatially separate the reserve and ready SV pools (Fergestad, 2001).

Previously characterized defects in exocytosis and endocytosis have suggested that SV maturation in stoned mutants may be impaired. Elegant studies on rat hippocampal cultures have recently estimated the time course for SV maturation ('repriming') to be from 5 to 40 sec. To test whether the delay period from endocytosis to exocytosis is increased in stoned mutants, the lapsed time required before loaded dye could be released from NMJ boutons was examined. FM1-43 dye was loaded with high-K+ saline (30 sec and 1 and 5 min), and then the preparation was washed in calcium-free saline for a variable period before K+-evoked unloading. No significant change between controls and stoned mutant boutons in the amounts of FM1-43 release was detected in these assays. These studies suggest that although fewer SVs are recycled via the endo-exo pool in stoned mutants, no difference in the rate of SV maturation is detectable (Fergestad, 2001).

Time-lapse studies using FM1-43 dye uptake assays indicate the rates of membrane retrieval after the fusion event to be t1/2 of ~20 sec or even faster. To determine whether endocytosis is delayed after exocytosis in stoned mutants FM1-43 was applied either during a 30 sec high-K+ stimulus or for 30 sec immediately after the stimulus. In wild-type NMJ terminals, a 30 sec application of high-K+ saline with FM1-43 loads synaptic terminals to levels similar to those of longer loading times, indicating that endocytosis is tightly temporally coupled to exocytosis. FM1-43 application for 30 sec immediately after the stimulation results in much lower levels of dye uptake, indicating that reduced endocytosis continues after the stimulation period. In contrast, stoned mutant boutons display greatly reduced endocytosis during the initial time period, when exocytosis and endocytosis levels are normally tightly coupled, and substantial levels of dye uptake only after the depolarizing stimulation. The striking dye uptake difference normally seen between stoned mutants and control animals is no longer present when the dye is added to the preparation after a 30 sec delay. Because longer dye application times do not allow complete loading, the delay in loading the stoned SV pool cannot alone account for the decreases in overall dye uptake. Thus, stoned mutants show both a significantly delayed onset of endocytosis and a significantly smaller recycling SV pool (Fergestad, 2001).

The Stoned proteins and Synaptotagmin specifically interact in the presynaptic terminal. Both StnA and StnB have been shown to bind Synaptotagmin directly, and Synaptotagmin is specifically mislocalized and subsequently degraded in stoned mutants. Moreover, the stoned and synaptotagmin mutant phenotypes are strikingly similar; both show comparably decreased and nonsynchronous synaptic transmission, decreased synaptic vesicle density, and aberrant, enlarged synaptic vesicles. One hypothesis to explain these diverse findings is that the Stoned proteins and Synaptotagmin mediate the same endocytotic function and that Stoned is required to recruit and/or maintain Synaptotagmin during plasma membrane endocytosis (Fergestad, 2001).

A key prediction of this hypothesis is that elevated levels of Synaptotagmin should alleviate the severe phenotypes observed in stoned mutants. To test this hypothesis, neurally expressing GAL4 drivers were used to mediate expression of a UAS-Synaptotagmin transgene construct, thus elevating Synaptotagmin levels in synaptic boutons. Whether overexpression of Synaptotagmin in the embryonic lethal stoned mutant background would rescue viability was first tested. Homozygous lethal stn13-120 animals, containing the UAS-Synaptotagmin construct alone, remain embryonic lethal in the absence of a GAL4 driver. However, two temporally different neural GAL4 drivers both rescue the embryonic lethality of stn13-120 in a manner consistent with the onset of their expression. (1) The 1407-GAL4 driver expresses throughout the nervous system during embryogenesis but ceases expression after hatching. When the 1407 driver is crossed to UAS-Synaptotagmin; stn13-120 animals, mutant animals now hatch (~98%) at normal times but then proceed to die as L1 stage larva, consistent with the termination of the 1407 expression. (2) The 4G-GAL4 driver is expressed during later stages of embryogenesis but then remains expressed in the nervous system throughout the life of the animal. When driving Synaptotagmin expression in stn13-120 mutants with the 4G driver, embryos also now hatch (~96%), although this hatching is delayed (mutant animals now hatch between 21 and 35 hr AF), consistent with the later onset of expression. Furthermore, maintained Synaptotagmin overexpression with 4G now rescues the embryonic lethal allele stn13-120 to adult viability. These results show that elevated Synaptotagmin can rescue the stoned lethality and that persistent elevated Synaptotagmin is required to compensate for the loss of Stoned during maintained synaptic function (Fergestad, 2001).

The prediction from these studies is that elevated levels of Synaptotagmin can alleviate the endocytosis defects caused by stoned mutation. To test this prediction, FM1-43 dye uptake was assayed at the larval NMJ. Overexpression of Synaptotagmin in stnC mutants rescues the endocytotic functional defects observed in stnC. UAS-Synaptotagmin; stnC larvae without a GAL4 driver are similar to stnC mutants alone and no rescue of the dye uptake defect is seen. Strikingly, however, the introduction of the 4G-GAL4 driver almost completely rescues the defects in dye uptake; no longer significantly different from control. Interestingly, the overexpression of Synaptotagmin alone in a control background (4G/UAS-Synaptotagmin) shows a striking increase in the amount of dye loaded, as compared with that of controls. These findings show that the stoned mutant phenotypes can be directly rescued by elevation of Synaptotagmin levels in the presynaptic terminal. The similarity of the stoned and synaptotagmin mutant phenotypes and the data presented here suggest that the sole role for the Stoned proteins may be to maintain the presynaptic function of Synaptotagmin (Fergestad, 2001).

The Drosophila stoned locus was identified 35 years ago based on severe behavioral impairments. It is one of the few dicistronic loci characterized in Drosophila, encoding Stoned A and B proteins; however, the Stoned A protein appears totally dispensable for known functions. In contrast, the crucial importance of the Stoned B protein for synaptic transmission has been well established. Nevertheless, the exact mechanistic function of STNB remains enigmatic. This study has generated and characterized a graded set of STNB hypomorphic animals to provide evidence that STNB has a dose-dependent limiting function regulating neurotransmission strength as a potent sorting factor governing a specific set of integral vesicular proteins during synaptic vesicle reconstitution. The key findings supporting this interpretation of STNB function are (I) the occurrence of diminished and altered release in a progressive series of STNB hypomorphic alleles, in the absence of significant ultrastructural defects of the vesicle pools, thereby indicating a changed fusion competence of synaptic vesicles, and (II) the differential loss of integral synaptic vesicle proteins dependent on the level of STNB activity (Mohrmann, 2008).

In previous studies, severely compromised basal synaptic transmission has been reported as a prominent feature of the physiological phenotype of various classical stoned mutants. Simplistically, defective synaptic release might be entirely a secondary effect of a primary impairment in vesicle pool maintenance, due to disrupted vesicle endocytosis. However, recent studies of endophilin and synaptojanin endocytic mutants suggest that only a surprisingly small number of synaptic vesicles is actually required to support normal synaptic function at basal stimulation frequencies: In both endophilin and synaptojanin mutants, basal synaptic transmission is completely normal despite the near elimination of the presynaptic vesicle population, and the loss of synaptic uptake of FM1-43. This raises the question that STNB may be involved in other processes that affect exocytosis, apart from limiting the availability of vesicles. The observation that viable stnC mutants exhibit release defects without major alterations in vesicle pool size seems to support such notion. However, recent findings show that the stnC mutation induces the expression of a C-terminally truncated STNB variant, together with low levels of the full-length product, thereby possibly involving dominant-negative effects or a partial functionality of the truncated STNB variant. Thus, the physiological phenotype in stnC mutants cannot be clearly interpreted. To clarify whether a hypomorphic condition exists that would allow for a segregation of the putative release defect and vesicle pool depletion, a new collection of graded STNB hypomorphic alleles was generated by transgenic expression of STNB in the stn13-120 mutant background. In the hypomorphic condition, STNB levels should be sufficient to support vesicle resupply and to maintain normal vesicle pools, but the shortage would still compromise basal synaptic transmission (Mohrmann, 2008).

This study demonstrates that physiological defects first emerge when STNB expression is reduced to less than 40% of wildtype level. Below this threshold, the decrease in basal EJC amplitude correlates closely with the expression level of STNB. Hypomorphic stn-vl mutants expressing ~35% of the wildtype level are of particular interest for this analysis, because their expression is only slightly lower than this threshold, and yet this condition causes a large 40% drop in basal amplitude. Strikingly, the ultrastructural analysis of stn-vl synapses showed that clustered and docked vesicle pools at the presynaptic active zone were completely unaffected in this hypomorphic condition, although there is a slight reduction in overall vesicle density. At the Drosophila NMJ, an 'exo/endo cycling vesicle pool' (ECP) has been demonstrated at the bouton periphery, which probably corresponds to the electrophysiologically defined readily releasable pool (RRP), and a reserve pool (RP) at the bouton center. According to this study the ECP/RRP alone is sufficient to allow for full-scale basal synaptic transmission after pharmacological depletion of the RP, and a loss of RP vesicles mainly affects synaptic fatigue during high-frequency stimulation. Since ultrastructural data indicate the integrity of the ECP/RRP in stn-vl hypomorphs, it must be concluded that the reduction in average amplitude during low frequency stimulation cannot be simply due to a small vesicle depletion restricted to the RP. Rather, defective basal synaptic transmission must be caused by the reluctant fusion of existing vesicles, correlating with the loss of STNB (Mohrmann, 2008).

In order to fully characterize this potential adverse effect on exocytosis, synaptic response patterns evoked by different stimulation paradigms were analyzed. Strikingly, stn-vl mutants exhibited significantly less pronounced synaptic depression during short stimulus trains applied at different frequencies. Interestingly, altered depression was found only in those low STNB-expressing hypomorphs that also showed defective basal transmission, suggesting a linkage relationship between these phenotypes. A simplistic model of synaptic depression could be satisfied by a progressive depletion of fusion-ready synaptic vesicles during phases of increased activity. Using shibire mutants to study depression in the absence of compensating endocytosis, it has been demonstrated that short trains (5-20Hz) that selectively deplete the readily-releasable pool caused a depression profile whose shape is reminiscent of the de-staining kinetics of FM1-43 labeled RRP in presynaptic boutons. Hence, the initial phase of depression at NMJ synapses presumably reflects the depletion of the RRP. Based on this interpretation, the altered depression profile in STNB hypomorphs is most likely due to an alteration in mobilization and/or fusion-rate of RRP vesicles. Since abnormal response patterns are also observed for simple paired-pulse stimuli, a shortage of STNB might generally change release properties. It is widely accepted that basal release probability is a determinant factor for short-term plasticity. Indeed, the presence of a depletion-based mechanism of synaptic depression readily implies a dependence of depression kinetics on initial release probability. Therefore, the reduction in basal EJC amplitude and the slowing of depression kinetics represent concurring indicators of an underlying decrease of release probability caused by removal of STNB (Mohrmann, 2008).

Though STNB could in principle play a dual role by independently functioning in exocytosis and endocytosis, no evidence was found to support such hypothesis. In fact, stn-vl hypomorphs, which exhibited less pronounced synaptic depression, also demonstrated a delayed recovery after prolonged stimulation indicating an accompanying defect in vesicle recovery. More likely, STNB serves functions on two different levels during vesicle reconstitution: Apart from simply being an essential component constituting functional endocytic complexes, STNB potentially also acts on a governing stage ensuring proper recovery of fusion-competent vesicles. Since a compromised complement of synaptic proteins on recovered vesicles could readily account for the decreased release probability in stnB hypomorphs, assays were performed for possible alterations in presynaptic expression levels and localization of synaptic vesicle proteins. While the expression of all tested proteins was at least slightly reduced in stn-vl hypomorphs, a definite subset of integral vesicle proteins was clearly most affected by STNB depletion. Synaptobrevin and synapsin expression levels were only slightly (≤30%) reduced, while the abundance of synaptotagmin and vGLUT were more severely decreased (≤50%). Synapsin adheres to available vesicular membranes in a dynamic fashion based on its phosphorylation state and presumably without obligatory endocytic sorting. Therefore, the reduction in synapsin levels likely represents a decrease in vesicle number within presynaptic terminals. Indeed, this conclusion is well supported by the comparable level of reduction in vesicle density observed at the ultrastructural level. However, the more pronounced effects on synaptotagmin and vGLUT cannot be attributed simply to a physical depletion of vesicles, suggesting a specifically reduced presence in vesicular membranes, consistent with trafficking defects (Mohrmann, 2008).

A mislocalization of synaptotagmin-1 was already reported in lethal stoned mutants, spawning discussions of a synaptotagmin-focused function of stoned proteins. New data shows that presynaptic expression of different vesicle proteins is differentially affected by reduced STNB levels, excluding the possibility that defective endocytosis causes an nonselective loss of vesicle proteins from synaptic boutons. The new data also suggest that STNB function might be important for the correct localization of a specified set of vesicle proteins, which questions whether the loss of synaptotagmin-1 alone is primarily responsible for the physiological phenotypes observed in stoned mutants. Based on the finding that stnB hypomorphs exhibit impaired neurotransmitter release, and accompanying alterations in vesicle protein configuration, it is considered most likely that STNB acts as a stabilizing and/or sorting factor for several synaptic vesicle proteins supporting this function. Similarly, a recent study (Diril, 2006) postulated that the mammalian STNB homolog stonin2 acts as an endocytic sorting adapter for synaptotagmin-1. Confusingly, however, STNB lacks several N-terminal WVxF motifs of its ortholog, which supposedly mediate a stonin2-AP2 interaction crucial for the reported stonin2-induced acceleration of synaptotagmin endocytosis. Nevertheless, association with other AP2-interacting proteins might also enable the recruitment of STNB to appropriate sites (Mohrmann, 2008).

Synaptotagmin sorting is predicted to critically depend on the ability to interact with the MHD of STNB. To examine the role of the MHD-synaptotagmin interaction, targeted mutations were generated to abolish this binding capability. Most interestingly, the complete removal of the MHD, and similarly the introduction of two point mutations within the putative binding interface for synaptotagmin, prevented the expressed STNB protein from restoring viability in lethal stoned mutants. Epitope-tagged mutant variant proteins could be neuronally expressed in wildtype animals, but completely failed to localize at synaptic sites. This suggests a crucial role for MHD-based interactions in presynaptic trafficking or anchorage of STNB. It is noteworthy that the STNB Y1125G, R1135A variant could still enter proximal parts of the axon, possibly indicating defective active transport of the protein. In contrast, the C-terminally truncated STNB variant is fully retained in discrete, punctate accumulations within the soma. Surprisingly, a similar, truncated variant of stonin2 seems to distribute uniformly in mammalian neurons (Walther, 2004). This might be due to higher expression levels, or different interactions of the remaining N-terminal portion of stonin2. A STNB-AP50 chimera, which contains corresponding sequences of AP50 instead of its original MHD, was also completely missing from synaptic sites, and exhibited punctate somatic localization similar to the truncated STNB variant. Thus, the MHD and the μ2-subunit are not functionally equivalent, even though the transplanted sequences contain the putative synaptotagmin-binding interface, and are predicted to confer the ability to bind synaptotagmin. Hence, STNB localization is not dependent on an association with synaptotagmin. This conclusion is independently confirmed by showing that synaptotagmin null mutants sytAD4 exhibit only relatively minor changes in STNB localization (Mohrmann, 2008).

Unlike the well-established SYT-STNB interaction, a direct binding activity between vGLUT and STNB has not been tested. Though the molecular mechanisms of STNB dependent vGLUT localization/stabilization are unclear, several recent studies in mammals report a direct interaction between its vertebrate homolog, VGLUT1, and endophilin, thereby establishing a connection to the endocytic protein network. It will be very interesting to examine the exact relationship between STNB and vGLUT in the future (Mohrmann, 2008).